Non-Immune Binding of Human IgG to M-Related Proteins Confers Resistance to Phagocytosis of Group A Streptococci in Blood

The non-immune binding of immunoglobulins by bacteria is thought to contribute to the pathogenesis of infections. M-related proteins (Mrp) are group A streptococcal (GAS) receptors for immunoglobulins, but it is not known if this binding has any impact on virulence. To further investigate the binding of immunoglobulins to Mrp, we engineered mutants of an M type 4 strain of GAS by inactivating the genes for mrp, emm, enn, sof, and sfbX and tested these mutants in IgG-binding assays. Inactivation of mrp dramatically decreased the binding of human IgG, whereas inactivation of emm, enn, sof, and sfbx had only minor effects, indicating that Mrp is a major IgG-binding protein. Binding of human immunoglobulins to a purified, recombinant form of Mrp indicated that it selectively binds to the Fc domain of human IgG, but not IgA or IgM and that it preferentially bound subclasses IgG₁>IgG₄>IgG₂>IgG₃. Recombinant proteins encompassing different regions of Mrp were engineered and used to map its IgG-binding domain to its A-repeat region and a recombinant protein with 3 A-repeats was a better inhibitor of IgG binding than one with a single A-repeat. A GAS mutant expressing Mrp with an in-frame deletion of DNA encoding the A-repeats had a dramatically reduced ability to bind human IgG and to grow in human blood. Mrp exhibited host specificity in binding IgG; human IgG was the best inhibitor of the binding of IgG followed by pig, horse, monkey, and rabbit IgG. IgG from goat, mouse, rat, cow, donkey, chicken, and guinea pig were poor inhibitors of binding. These findings indicate that Mrp preferentially binds human IgG and that this binding contributes to the ability of GAS to resist phagocytosis and may be a factor in the restriction of GAS infections to the human host.     

Lysophospholipid-Containing Membranes Modulate the Fibril Formation of the Repeat Domain of a Human Functional Amyloid,Pmel17

Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein–lysolipid interactions within melanosomes may regulate amyloid formation in vivo.     

Identification of a Novel Human Gene Containing the Tetratricopeptide Repeat Domain from the Down Syndrome Region of Chromosome 21

The Down syndrome (DS) region on chromosome 21, which is responsiblefor the DS main features, has been defined by analysis of DSpatients with partial trisomy 21. Within the DS region, we constructeda 1.6-Mb P1 contig map previously. To isolate gene fragmentsfrom the 1.6-Mb region, we performed direct cDNA library screeningand exon trapping using the P1 clones and a human fetal braincDNA library, and obtained 67 cDNA fragments and 52 possibleexons. Among them, 23 cDNA fragments and 4 exons were interpretedto be derived from a single gene by localization on P1 clonesand by Northern analysis. To obtain the full-length cDNA sequence,longer cDNA clones were further screened from another humancDNA library which was enriched with longer cDNA species. Theseclones were sequenced and assembled to a sequence of 9045 bp.This transcribed sequence encodes a novel 2025 amino-acid proteincontaining tetratricopeptide repeat (TPR) motifs and thereforethe gene was designated as TPRD (a gene containing the TPR motifson the Down syndrome region). The TPR domain has been foundin a certain protein phosphatase and in other proteins involvedin the regulation of RNA synthesis or mitosis. The TPRD gene,the novel gene which was proved to be in the 1.6-Mb region andto have the interesting features described above, is a candidatefor genes responsible for the DS phenotypes.     

Structural and Biochemical Consequences of Disease-Causing Mutations in the Ankyrin Repeat Domain of the Human TRPV4 Channel

The TRPV4 calcium-permeable cation channel plays important physiological roles in osmosensation, mechanosensation, cell barrier formation, and bone homeostasis. Recent studies reported that mutations in TRPV4, including some in its ankyrin repeat domain (ARD), are associated with human inherited diseases, including neuropathies and skeletal dysplasias, probably because of the increased constitutive activity of the channel. TRPV4 activity is regulated by the binding of calmodulin and small molecules such as ATP to the ARD at its cytoplasmic N-terminus. We determined structures of ATP-free and -bound forms of human TRPV4-ARD and compared them with available TRPV-ARD structures. The third inter-repeat loop region (Finger 3 loop) is flexible and may act as a switch to regulate channel activity. Comparisons of TRPV-ARD structures also suggest an evolutionary link between ARD structure and ATP binding ability. Thermal stability analyses and molecular dynamics simulations suggest that ATP increases stability in TRPV-ARDs that can bind ATP. Biochemical analyses of a large panel of TRPV4-ARD mutations associated with human inherited diseases showed that some impaired thermal stability while others weakened ATP binding ability, suggesting molecular mechanisms for the diseases.     

Structure of Human MDM4 N-Terminal Domain Bound to a Single-Domain Antibody

The N-terminal domain of MDM4 binds to the N-terminal transactivation domain of the tumor suppressor p53 and is an important negative regulator of its transactivation activity. As such, inhibition of the binding of MDM4 to p53 is a target for anticancer therapy. The protein has not been crystallized satisfactorily for structural studies without the addition of an N-terminal p53 peptide. We selected a single-domain antibody (VH9) that bound to the human domain with a dissociation constant of 44 nM. We solved the structure of the complex at 2.0-Å resolution. The asymmetric unit contained eight molecules of VH9 and four molecules of MDM4. A molecule of VH9 was located in each transactivation domain binding site, and the four non-MDM4-bound VH9 domains provided additional crystal contacts. There are differences between the structures of human MDM4 domain bound to VH9 and those of human and zebra fish MDM4 bound to a p53 peptide. Molecular dynamics simulations showed that the binding pocket in the three MDM4 structures converged to a common conformation after removal of the ligands, indicating that the differences are due to induced fit. The largest conformational changes were for the MDM4 molecules bound to p53. The simulated and observed structures should aid rational drug design. The use of single-domain antibodies to aid crystallization by creating a molecular scaffold may have a wider range of applications.     

Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva

Salivaricin A (SalA), the first Streptococcus salivarius lantibiotic to be characterized, appears to be inhibitory to most Streptococcus pyogenes strains. A variant of the SalA structural gene (salA1) is present in more than 90% of S. pyogenes strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (salA2 to salA5) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against Micrococcus luteus and in their ability to act as inducers of SalA production. It had previously been shown that S. pyogenes strain SF370 had a deletion (of approximately 2.5 kb) in the salM and salT genes of the salA1 locus. In the present study, several additional characteristic deletions within the salA1 loci were identified. S. pyogenes strains of the same M serotype all share the same salA1 locus structure. Since S. salivarius is a predominant member of the normal oral flora of healthy humans, strains producing anti-S. pyogenes lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing S. salivarius or who have been colonized with the SalA2-producing probiotic S. salivarius K12.     

Inhibition of Human Dyskerin as a New Approach to Target Ribosome Biogenesis

The product of the DKC1 gene, dyskerin, is required for both ribosome biogenesis and telomerase complex stabilization. Targeting these cellular processes has been explored for the development of drugs to selectively or preferentially kill cancer cells. Presently, intense research is conducted involving the identification of new biological targets whose modulation may simultaneously interfere with multiple cellular functions that are known to be hyper-activated by neoplastic transformations. Here, we report, for the first time, the computational identification of small molecules able to inhibit dyskerin catalytic activity. Different in silico techniques were applied to select compounds and analyze the binding modes and the interaction patterns of ligands in the human dyskerin catalytic site. We also describe a newly developed and optimized fast real-time PCR assay that was used to detect dyskerin pseudouridylation activity in vitro. The identification of new dyskerin inhibitors constitutes the first proof of principle that the pseudouridylation activity can be modulated by means of small molecule agents. Therefore, the presented results, obtained through the usage of computational tools and experimental validation, indicate an alternative therapeutic strategy to target ribosome biogenesis pathway.     

神经系统富亮氨酸重复超家族成员LRRN3 C端结构域蛋白的原核表达和纯化

神经系统富亮氨酸重复超家族成员LRRN3是一种重要的膜蛋白,与神经系统发生发育和损伤后修复密切相关．运用多聚酶链式反应（PCR）方法,获得长555bp的DNA序列,扩增产物克隆至pET21质粒中,构建Mal和His融合表达质粒,在大肠杆菌中诱导表达融合蛋白,经Ni^＋-NTA agarose亲和层析纯化获得融合蛋白,并以Western blotting鉴定,为进一步研究LRRN3基因的结构和功能奠定了基础．     

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.     

The Heptad Repeat 2 Domain Is a Major Determinant for Enhanced Human Immunodeficiency Virus Type 1 (HIV-1) Fusion and Pathogenicity of a Highly Pathogenic HIV-1 Env

Human immunodeficiency virus type 1 (HIV-1)-mediated depletion of CD4⁺ lymphocytes in an infected individual is the hallmark of progression to AIDS. However, the mechanism for this depletion remains unclear. To identify mechanisms of HIV-1-mediated CD4 T-cell death, two similar viral isolates obtained from a rapid progressor patient with significantly different pathogenic phenotypes were studied. One isolate (R3A) demonstrates enhanced pathogenesis in both in vivo models and relevant ex vivo lymphoid organ model systems compared to another isolate, R3B. The pathogenic determinants were previously mapped to the V5-gp41 envelope region, correlating functionally with enhanced fusion activity and elevated CXCR4 binding affinity. To further elucidate specific differences between R3A and R3B within the V5-gp41 domains that enhance CD4 depletion, R3A-R3B chimeras to study the V5-gp41 region were developed. Our data demonstrate that six residues in the ectodomain of R3A provide the major determinant for both enhanced Env-cell fusion and pathogenicity. Furthermore, three amino acid differences in the heptad repeat 2 (HR-2) domain of R3A determined its fusion activity and significantly elevated its pathogenic activity. The chimeric viruses with enhanced fusion activity, but not elevated CXCR4 affinity, correlated with high pathogenicity in the thymus organ. We conclude that the functional domain of a highly pathogenic HIV-1 Env is determined by mutations in the HR-2 region that contribute to enhanced fusion and CD4 T-cell depletion.Human immunodeficiency virus type 1 (HIV-1) is the causative agent for AIDS, which is characterized by a dramatic loss of CD4⁺ lymphocytes and impairment of the immune system against invading pathogens (13, 21, 22). Though much has been determined regarding interactions between HIV-1 virus and CD4⁺ target cells, the mechanisms by which the HIV-1 virus depletes CD4⁺ lymphocytes remain incompletely understood. Various studies have demonstrated that in an HIV-infected host, both infected and uninfected cells are prone to destruction, albeit by different pathways (15, 18, 29). Recently, our group and others have shown that while binding of CD4 and chemokine receptors contribute to syncytium formation in vitro, viral membrane fusion by the envelope glycoprotein plays an important role in depletion of both uninfected and infected cells by HIV-1 and simian-human immunodeficiency virus in vivo (1, 11, 12, 26, 29).HIV-1 entry into a cell is mediated by a multistep process that begins with high-affinity binding between viral envelope (gp120) and the cellular CD4 receptor (9, 14, 16). This binding causes a conformational change in the viral envelope, allowing for subsequent coreceptor binding (mainly CCR5 or CXCR4). Upon coreceptor binding, another conformational change is thought to take place that allows gp41 to engage the cell to form a fusion complex. Envelope proteins have been demonstrated to exist as a trimer, allowing for three gp41s to form a fusion assembly through noncovalent interactions. This fusion assembly is determined to exist in a six-helix bundle formation as the fusion event takes place, allowing for the virion to fuse to the host cell (5, 24).The envelope glycoprotein (Env) of HIV plays a significant role in viral pathogenesis, as seen in several in vitro and in vivo models of infection. The Env functions to mediate virus entry of cells and is also a major target for immune responses (31, 39). While the envelope initially forms as a precursor protein (gp160), subsequent cleavage by a cellular protease yields the surface subunit gp120 and the transmembrane gp41 although the gp120 and gp41 interact noncovalently (36). The gp120 protein is comprised of five variable (V1 to V5) and five conserved constant (C1 to C5) domains and binds CD4 and the coreceptors. The gp41 protein is comprised of an amino-terminal fusion domain and two heptad repeats (HR-1 and HR-2) in the ectodomain (extracellular domain), a single transmembrane domain, and a cytoplasmic tail (intracellular domain) (8, 10, 36, 37). Due to the discovery of fusion inhibitor peptides such as C34 (23, 24) and T20 (38), much is now known about the fusion complex formed by the HIV-1 fusion domain. Similar to other viral envelopes that carry a type 1 fusion complex (such as influenza and corona viruses), the ectodomain of HIV-1 Env carries two HRs that form a coiled-coiled structure. In order for HIV-cell fusion to occur, the HR-1 domains of the trimeric Env protein must interact with the cell surface. Following this initial interaction, HR-2 domains are thought to intertwine over the HR-1 coils to form a stable six-helix bundle, which represents the gp41 core structure. X-ray crystallographic studies show that the six-helix bundle core consists of the HR-1 and HR-2 peptides bound in an antiparallel manner. This structure brings the fusion peptide to the target cell membrane, allowing for the formation of a fusion pore and the entry of virions into the cell.HIV-1 Env expressed on the surface of infected cells can induce cell-cell fusion with adjacent uninfected cells to form multinucleated syncytia and single cell lysis in cell culture and apoptosis in primary cells. Various models (both ex vivo and in vivo) have been utilized to study HIV-1-induced depletion of CD4⁺ lymphocytes. Models such as SCID-human thymus-liver (SCID-hu thy/liv), tonsil histoculture, and human fetal thymus organ culture (HFTOC) have demonstrated significant use in the study of acute infection and pathogenesis in the appropriate lymphoid organ microenvironment as they retain the organ structure and do not require exogenous stimulation for productive viral infection to occur (2, 20, 28, 32). More importantly, tissue culture-adapted HIV-1 isolates such as HXB2 fail to replicate in the SCID-hu thy/liv or HFTOC models (30, 33). Organ models such as the SCID-hu thy/liv and HFTOC thus more accurately demonstrate infection, replication, and pathogenicity of primary HIV-1 strains.Here, HFTOC is used to investigate mechanisms by which an HIV-1 virus with a highly pathogenic viral Env is able to deplete CD4⁺ lymphocytes. Two viral isolates obtained from rapid progressor patient 3 of the ALIVE cohort (40) show significant sequence homology, particularly in the Env region, while they carry stark differences in pathogenic ability (26, 27). One isolate (denoted R3A) was found to demonstrate enhanced fusion in cell-cell fusion assays as well as enhanced pathogenesis in relevant ex-vivo/in vivo organ model systems compared to another isolate, R3B. To define the pathogenic determinants that differentiate R3A from R3B, this study demonstrates that the enhanced fusogenicity of R3A (governed by the ectodomain of the gp41), but not the elevated CXCR4 binding affinity, confers the pathogenic phenotype in HFTOC. We further demonstrate that three amino acid differences in the HR-2 domain allow for this enhanced fusion for R3A Env, defining a possible mechanism for a pathogenic HIV-1 envelope.     

Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine.